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Illumina GAIIx for high throughput sequencing

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Illumina GAIIx for high throughput sequencing

  1. 1. Illumina Genome Analyzer IIx for high throughput sequencing Cosentino Cristian, PhD Genomics and Bioinformatics unit Filarete Foundation – Milan (IT) cosentia@gmail.com 15.4.2010
  2. 2. Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx Genome Analyzer IIx 3 Data management 4 Target enrichment
  3. 3. Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
  4. 4. Approaching to NGS 1 2010 2000 1980 1990 1977 Sanger sequencing method by F. Sanger (PNAS ,1977, 74: 560-564) 1983 PCR by K. Mullis (Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73) 1953 Discovery of DNA structure Human Genome Project (Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31) (Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351) 1993 Development of pyrosequencing (Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365) Single molecule emulsion PCR 1998 Founded Solexa 1998 Founded 454 Life Science 2000 454 GS20 sequencer 2005 (First NGS sequencer) Solexa Genome Analyzer 2006 (First short-read NGS sequencer) Illumina acquires Solexa 2006 (Illumina enters the NGS business) ABI SOLiD 2007 (Short-read sequencer based upon ligation) Roche acquires 454 Life Sciences 2007 (Roche enters the NGS business) GS FLX sequencer 2008 (NGS with 400-500 bp read lenght) NGS Human Genome sequencing 2008 (First Human Genome sequencing based upon NGS technology) Hi-Seq2000 2010 (200Gbp per Flow Cell)
  5. 5. Next-generation sequencing platforms 1 Isolation and purification of target DNA Sample preparation Clonally amplified DNAs Single molecule Amplific. Cluster generation Emulsion PCR on solid-phase Chemistry Sequencing by synthesis Sequencing by synthesis with reversible Pyrosequencing Sequencing by ligation with reversible terminators termintors Data analysis Illumina GAII Roche 454 ABi SOLiD Helicos HeliScope
  6. 6. Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
  7. 7. Illumina GAII GAIIx instruments 2 Bioanalyzer 2100 Flow Cell Cluster station Genome Analyzer IIx Paired-end module Linux server
  8. 8. Illumina GAII Applications 2 de novo sequencing (whole-genome) re-sequencing (whole-genome or targeted) RNA-seq smallRNA-seq CHiP-seq Single-read Paired-end Multiplexing
  9. 9. Illumina GAII Sequencing by synthesis with reversible terminator 2 Parameter Performance Amplification Bridge-PCR on solid-phase Chemistry SBS with reversible terminators Cost 2 $/Mbp Advantages Disadvantages •Most widely used platform (> 90 •Low multiplexing capability science/nature publication) •Substitution errors •Sample preparation automatable •SBS, real-time analysis and base calling are performed simultaneously to the run •Automated cluster generation procedure
  10. 10. Illumina GAII Coverage estimation 2 Sample 10000 preparation Clusters amplification 1000 Sequencing by synthesis Analysis 35 cycles pipeline 100 50 cycles 75 cycles Coverage/lane 100 cycles 10 35x2 cycles 50x2 cycles 75x2 cycles 1 100x2 cycles 0.001 0.01 0.1 1 10 0.1 Input DNA (Gbp)
  11. 11. Illumina GAII GAIIx sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
  12. 12. Illumina GAII Library preparation 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline gDNA Fragmented Adaptor- Gel purification DNA ligated DNA
  13. 13. Illumina GAII Library validation 2 Sample preparation gDNA Clusters amplification Sequencing by synthesis Analysis pipeline smallRNA Total RNA Bioanalyzer 2100
  14. 14. Illumina GAII GAIIx sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
  15. 15. Illumina GAII Flow cell 2 Sample Lanes preparation 1234 5678 Clusters amplification Sequencing by synthesis 120 Analysis tiles/lane pipeline
  16. 16. Illumina GAII Cluster generation 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Sample preparation Amplification Cluster Linearization, amplification blocking and primer hybridization SBS sequencing
  17. 17. Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Hybridize adapter-ligated forward fragment and extend Extension is completed Denature dsDNA and wash original forward template; reverse template stays covalently attached to the array
  18. 18. Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Bridge amplification of the reverse fragment Double-strand bridge is formed Double strand bridge is denatured and reverse as wel as forward fragments are covalently attached to the array
  19. 19. Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Bridge amplification is repeated to enlarge the cluster Double-strand bridges are denatured Reverse strands fragments are cleaved and washed away
  20. 20. Illumina GAII Bridge amplification 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Cluster with forward strands only, covalently attached to the array Sequencing primers start the SBS process
  21. 21. Illumina GAII Sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
  22. 22. Illumina GAII SBS technology 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline
  23. 23. Illumina GAII GAIIx optical path 2 Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Two colour excitation Four colour emission detection
  24. 24. Illumina GAII Single-read sequencing workflow 2 Sample preparation Clusters Cluster amplified amplification FlowCell Sequencing by synthesis Analysis pipeline Install prism Install flow-cell Apply oil First-base incorporation Adjust focus Check quality metrics 36-100 cycles sequencing run for Read 1 Read 1 analysis pipeline
  25. 25. Illumina GAII Paired-end sequencing workflow 2 Sample preparation Cluster amplified Clusters FlowCell amplification Sequencing by Install prism synthesis Analysis pipeline Install flow-cell Apply oil First-base incorporation Adjust focus Check quality metrics 36-100 cycles sequencing run Prepare Read 2 for Read 1 36-100 cycles sequencing run for Read 2 Read 1 and 2 analysis pipeline
  26. 26. Illumina GAII Paired-end strategy 2 Sample preparation Paired-end sequencing works into GA and uses chemicals from PE Clusters module to perform cluster amplification of the reverse strand amplification Sequencing by synthesis Analysis Single-read pipeline (CS) Paired-end (PEM)
  27. 27. Illumina GAII Paired-end strategy 2 Sample preparation Clusters Single-read (read 1) amplification Mapped Unmapped Sequencing by read read synthesis Reference Gene 1 Gene 2 sequence Analysis pipeline Sequence reads Paired-end (read 1 & read 2) Mapped Mapped read read Reference Gene 1 Gene 2 sequence Sequence reads
  28. 28. Illumina GAII Sequencing workflow 2 Sample Workbench preparation Clusters Cluster Station amplification Sequencing by Genome Analyzer synthesis Analysis pipeline Linux Server
  29. 29. Illumina GAII Firecrest and CASAVA 2 Sample Image files preparation Intensity files Base calls files Clusters amplification Firecrest Bustard From image From intensity Sequencing by synthesis to intensity to reads Analysis pipeline Gerald/ELAND Alignment to genome Assembly Alignment files GenomeStudio Data visualization CASAVA Consensus assembly Sequence ANALYSIS
  30. 30. Illumina GAII GenomeStudio viewer 2 Sample preparation Clusters amplification Small RNA sequencing Sequencing by synthesis Analysis pipeline
  31. 31. Illumina GAII GenomeStudio viewer 2 Sample preparation Clusters amplification Sequencing by synthesis RNA sequencing Analysis pipeline
  32. 32. Illumina GAII GenomeStudio viewer 2 Sample preparation Clusters amplification Sequencing by synthesis DNA sequencing Analysis pipeline
  33. 33. Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
  34. 34. High throughput data storage 3 Genotyping units 0.5 – 14 GB/Chip Tape recording unit Data storage for offline backup management server Sequencing unit 250 Tb storage capacity 1 Gbit LAN dedicated 1 – 6 Tb/FlowCell
  35. 35. High throughput data analysis 3 External services Sequencing pipeline Database server Genotyping applications Component Availability CPU 40 core equivalent RAM 1 Tb Storage on-line (HD) 250 Tb Storage off-line (Tape) 80 Tb normal 160 Tb compressed
  36. 36. Summary 1 Towards NGS sequencing 2 NGS with Illumina GAIIx 3 Data management 4 Target enrichment
  37. 37. High throughput sample preparation 4 Locus lenght or #loci Nature Methods, 2010, 7: 111-118
  38. 38. SureSelect target enrichment 4 Agilent SureSelect Solution-phase capture with streptavidin-coated magnetic beads Reported 60-80% of capture efficiency
  39. 39. The end Cosentino Cristian, PhD – cosentia@gmail.com

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